Field of the Invention
The present invention relates to the fields of molecular biology and medicine. More specifically, the invention relates to nucleic acids for use as DNA vaccines, and methods of using them to treat subjects suffering from or susceptible to allergic reactions.
Discussion of Related Art
Allergy is a hypersensitivity disease characterized by the production of IgE antibodies against an allergen, or allergy-causing molecule. Allergies affect more than 25% of the population. Allergens can enter the body through many routes, including the respiratory tract, skin contact, ingestion, insect bite, or injection of a drug.
Allergy disease management comprises diagnosis and treatment. Allergists diagnose an allergy using a variety of techniques, such as a skin prick test, radioallergosorbent-based techniques, ELISA, or provocation test to demonstrate allergen specific IgE and to identify the allergen source. Treatment of allergy most often falls into two categories: avoidance and dosing with anti-histamines. A third alternative, allergy immunotherapy, requires that the patient receive weekly injections consisting of small amounts of the offending allergens in order to help the immune system reeducate its response to the allergen.
The use and generation of allergen fusion proteins are well known in the art. For example, U.S. Pat. No. 7,566,456 teaches a fusion protein with IgE and IgG binding domains as well as encoding an allergen. Further, WO 97/07218 teaches allergen-anti-CD32 fusion proteins for use in allergy immunotherapy. Neither of these documents, however, teaches how their respective fusion protein interacts with T cells through antigen presentation to induce or modify a Th1 response. Furthermore, there is no theoretical connection between directing the anti-CD32 containing vaccine to dendritic cells to effect a positive induction of Th1 cells. Both of these documents teach a composition that introduces an allergen therapeutically, such that the allergen can be found in the serum as an allergen-fusion protein.
It has been established by Toda et al., 2002 that a T cell epitope of an allergen, in this case a Cry J2 epitope located at amino acid 247-258, can be attached to a fusion protein and be used to conduct allergy-specific immunotherapy. The specific composition described by Toda et al., 2002 is the use of a DNA vaccine encoding the major CD4 T cell epitope of Cry J2, located at amino acids 247-258, attached to class II-associated invariant chain peptide (CLIP). CLIP contains a lysosomal/endosomal trafficking sequence and contains a domain that binds to the peptide binding groove of MHC II. Toda et al., 2002 shows that immunization with the Cry J2 peptide/CLIP DNA vaccine results in priming a mouse to a predominantly Th1 response, characterized by higher IFN-gamma and IgG2a production. However, Toda et al. does not teach the intracellular targeting of the entire protein coding sequence of an allergen useful for conducting allergy-specific immunotherapy.
U.S. Pat. No. 6,982,326 and U.S. Pat. No. 6,090,386 describe nucleic acid sequences coding for the Cryptomeria japonica major pollen allergens Cry J1, Cry J2, Jun s I, and Jun v I, and fragments or peptides thereof. The invention also provides purified Cry J1, Cry J2, Jun s I, and Jun v I, and at least one fragment thereof produced in a host cell transformed with a nucleic acid sequence coding for Cry J1, Cry J2, Jun s I, and Jun v I, or at least one fragment thereof, and fragments of Cry J1, Cry J2, Jun s I, or Jun v I, or at least one fragment thereof, and fragments of Cry J1, Cry J2, Jun s I, or Jun v I prepared synthetically. Cry J1, Cry J2, Jun s I, and Jun v I, and fragments thereof are disclosed as useful for diagnosing, treating, and preventing Japanese cedar pollinosis. The invention also provides isolated peptides of Cry J1 and Cry J2. Peptides within the scope of the invention comprise at least one T cell epitope, or preferably at least two T cell epitopes of Cry J1 or Cry J2. The invention also pertains to modified peptides having similar or enhanced therapeutic properties as the corresponding naturally-occurring allergen or portion thereof but having reduced side effects. Methods of treatment or of diagnosis of sensitivity to Japanese cedar pollens in an individual and therapeutic compositions, and multi-peptide formulations comprising one or more peptides of the invention are also provided. The invention does not teach how to combine the epitopes or allergens into a DNA vaccine with immunostimulatory properties.
U.S. Pat. No. 7,547,440 and U.S. Pat. No. 7,112,329 identify the T-cell epitope site on a Japanese cypress (hinoki) pollen allergen molecule by stimulating a T-cell line established from a patient suffering from Japanese cypress pollen allergy with an overlap peptide covering the primary structure of the Japanese cypress pollen allergen. The peptide is useful in peptide-based immunotherapy for patients with spring tree pollinosis including patients with Japanese cypress pollinosis having cross reactivity with Japanese cypress pollen. The peptide is also useful for diagnosing spring tree pollinosis. The invention is limited to diagnostics and polypeptide delivery of epitopes.
DNA vaccines have been developed as an alternative to traditional whole cell or whole virus vaccines. Generally speaking, DNA vaccines are engineered nucleic acids that include sequences encoding one or more epitopes. The nucleic acids are delivered to cells, typically antigen presenting cells (APCs), the nucleic acids are expressed, and the epitopes present on the expressed proteins are processed in the endosomal/lysosomal compartment, and ultimately presented on the surface of the cell. U.S. Pat. No. 5,633,234 to August et al. discloses and characterizes the endosomal/lysosomal targeting sequence of the lysosomal-associated membrane protein (LAMP). This patent identifies critical residues in the C-terminal region of the protein, which are necessary for targeting of the protein to the endosomal/lysosomal compartment. The patent discloses that fusion of antigenic peptides to the C-terminal LAMP targeting sequence can provide enhanced processing and presentation of epitopes for generation of an immune response.
In addition, U.S. patent application publication number 2004/0157307 to Harris et al. discloses the use of the LAMP lumenal domain as a “trafficking domain” to direct chimeric proteins expressed from DNA vaccines through one or more cellular compartments/organelles, such as through the lysosomal vesicular pathway. The chimeric proteins include the lumenal domain of a LAMP polypeptide, an antigenic domain comprising a peptide epitope sequence previously identified and selected from an antigen protein, a transmembrane domain, and an endosomal/lysosomal targeting sequence.
DNA vaccines have been proposed as a treatment of allergic disease (Raz et al., 1996; Hartl et al., 2004; Hsu et al., 1996; Crameri 2007; Weiss et al., 2006). The underlying rationale is that allergen protein encoded by a DNA vaccine will preferentially activate the allergen-specific Th1 cellular response with the production of interferons by APCs, natural killer (NK), and T cells, rather than the characteristic Th2-type response, such as secretion of IL-4, IL-5, and IL-13, and the formation of IgE by B lymphocytes and the maturation and recruitment of eosinophils in late-phase reactions. However, the mechanisms underlying the differential induction of the Th1 and Th2 T-cell phenotypes appear to involve a large number of factors, such as unique properties of the bacterial DNA of vaccine preparations, e.g., unmethylated and CpG DNA residues, the cytokine milieu elicited by innate immunity, and the cellular trafficking properties of the allergens (Chen et al., 2001; Kaech et al., 2002). No invention or method has successfully addressed the uncertainty of allergy treatment as conducted by delivery of nucleic acids encoding an allergen. Thus, to date such a method of allergy treatment has not been enabled. In addition, administration of DNA vaccines for the treatment of allergic disease has resulted in the secretion of the allergen peptide into the extracellular environment, potentially leading to accidental induction of an allergic response through activation of IgE.